A non-natural amino acid-incorporated protein (alloprotein) which has a substitution of an amino acid residue at a desired position by an amino acid (a non-natural amino acid) other than 20 kinds of amino acids involved in normal protein synthesis could be an effective measure of analyzing the structure and function of a protein. Over 30 kinds of alloproteins have already been synthesized using aminoacyl-tRNA synthetase (aaRS)/tRNA pair derived from various biological species. A system which has most long history and is applied to incorporation of a lot of useful non-natural amino acids is a pair of a tyrosyl-tRNA synthetase (TyrRS) mutant and an amber-suppressed tRNATyr. In this method, the following orthogonal relationship makes a key point: each of aaRSs in two groups of eubacteria and of archaebacteria and eukaryotes may aminoacylate tRNA in its group, whereas it could not aminoacylate tRNAs in the other group. For example, the TyrRS/tRNATyr pair of archaebacterium Methanocaldococcus jannaschii is an orthogonal pair in E. coli system, whereas the pair of Escherichia coli TyrRS and Bacillus stearothermophilus tRNATyr is an orthogonal pair in mammalian cell system. Therefore, these pairs may be used for extending genetic code in their systems (see, for example, Patent Document 1 and Non-Patent Document 1).
On the other hand, Methanosarcina mazei-derived pyrrolysyl-tRNA synthetase (PylRS) and amber suppressor tRNAPyl function as orthogonal aaRS/tRNA pair in E. coli cells (see, for example, Non-Patent Document 2). Furthermore, it is reported that this pair may also be used for extending genetic code in eukaryotic cell (see, for example, Patent Document 2). Pyrrolysine is a lysine derivative having a bulky methylpyrroline moiety at the side chain. Wild-type PylRS may bind Nε-Boc-L-Lysine to tRNAPyl in E. coli cells (see Patent Document 2). Moreover, X-ray crystal structure of a complex of wild-type PylRS, ATP analog, and pyrrolysine or pyrrolysine analog is reported (see Non-Patent Documents 3, 4 and 9).    [Patent Document 1] WO2004/070024    [Patent Document 2] Japanese Patent Kokai Publication No. JP-P2007-37445A    [Non-Patent Document 1] Sakamoto, K. et al., Nucleic Acids Research, 2002, Vol. 30, pp. 4692-4699.    [Non-Patent Document 2] Blight S. K. et al., Nature, (2004) Vol. 431, pp. 333-335.    [Non-Patent Document 3] Yanagisawa, T. et al., Acta Cryst. (2006) F62, 1031-1033    [Non-Patent Document 4] Kavran, J. M. et al., Proc. Natl. Acad. Sci. (2007) Vol. 104, pp. 11268-11273    [Non-Patent Document 5] Tsao, M.-L., Tian, F., Schultz, P. G. ChemBioChem Vol. 2005, Issue 6, pp. 2147-2149    [Non-Patent Document 6] Ohno, S. et al., J. Biochem. (Tokyo) Vol. 141, pp. 335-343 (2007)    [Non-Patent Document 7] Mukai, et al., Biochem. Biophys. Res. Commun. Vol. 371, pp. 818-822 (2008)    [Non-Patent Document 8] Liu, W. et al., Nat. Methods. Vol. 4, pp. 239-244 (2007)    [Non-Patent Document 9] Yanagisawa, T. et al., J. Mol. Biol. (2008) 378, 634-652